1. Field of the Invention
The present invention relates to a nucleic acid purification method and purification apparatus and particularly to the purification method and purification apparatus suited to separate nucleic acid contained in the biological sample from the coexisting substance and to take it out.
2. Description of the Prior Art
A great number of gene technologies have been developed due to progress of molecular biology, and many pathetic genes have been separated and identified by such technologies. As a result, In the field of medical treatment as well, a technique of molecular biology is adopted in the diagnosis or inspection method. This makes it possible to perform the diagnosis which is extremely difficult so far and to achieve a substantial reduction in the number of days for inspection.
Such a progress is attributable to commercial application of gene amplification method, especially, polymerase chain reaction (called PCR method) in many respects. The PCR method makes it possible to amplify nucleic acid in the solution sequence-specifically. For this reason, it indirectly proves existence of a very small number of viruses present in the serum, by amplifying and detecting the nucleic acid as genes of these viruses. However, there are some problems when this PCR method is used for clinical applications in daily examinations. Especially, extraction of nucleic acids in the pretreatment process and purification process are important to maintain accuracy. Some techniques have been proposed regarding purification of nucleic acid.
According to the method disclosed in Japanese Official Patent Gazette 266864/1999, a nucleic acid capturing tip incorporating solid phase containing silica is used to extract nucleic acid automatically. A nozzle tip is mounted on the liquid suction/discharge movable nozzle and sucks from the bottle the binding enhancer to accelerate nucleic acid to be bound onto said solid phase. Then it sucks the sample containing nucleic acid from the specimen vessel and discharge their mixture into the reaction vessel. After the mixture has been discharged, the nozzle tip is discarded, and a new nucleic acid capturing tip is mounted in position. The mixture is sucked from inside said reaction vessel and discharged into the nucleic acid capturing tip connected to the liquid suction/discharge movable nozzle. Then nucleic acid in the sucked mixture is bound with the solid phase in the nucleic acid capturing tip, and liquid in said nucleic acid capturing tip is discharged. Then washing solution discharged in the washing vessel is sucked into said nucleic acid capturing tip, and said washing solution is discharged from said nucleic acid capturing tip. Said solid phase bound with nucleic acid and interior of said nucleic acid capturing tip are washed. Further, eluent is sucked into the said nucleic acid capturing tip after having been washed, and eluent containing the nucleic acid separated from said solid phase is discharged into a vessel for purified product.
According to the method disclosed in Japanese Official Patent Gazette 266864/1999, when there is reaction between the sample containing nucleic acid and various types of reagents, the sample containing nucleic acid is sucked from the specimen vessel or reaction vessel and is dispensed into the targeted reaction vessel. So said sample may scatter up to the mechanism holding the nozzle tip, and may cause contamination which will give a serious influence to nucleic acid purifying performances.
The solid phase bound with nucleic acid and the interior of the nucleic acid capturing tip are washed. The washing solution discharged into special-purpose vessel is sucked into said tip and is discharged into the special-purpose vessel. This process is repeated, resulting in a poor cleaning efficiency. Further, if washing solution remains in the solid phase of said tip, the next eluent concentration will be affected to deteriorate nucleic acid purifying performances.
The object of the present invention is to provide a nucleic acid purification method and purification apparatus which ensures excellent washing efficiency without causing contamination or liquid remaining in the nozzle tip.
To achieve the above object, the following means are provided:
In a nucleic acid purification method wherein a nucleic acid capturing tip incorporating the solid phase containing a nucleic acid capturing agent is used to allow said solid phase to capture a nucleic acid and to extract the nucleic acid, the present invention is characterized in that washing solution is fed in the tip containing the solid phase capturing said nucleic acid unidirectionally from the head to the end.
Silica can be cited as an specific example of said nucleic acid capturing agent. Fine particles mainly composed of silica, fibers and fibrous wool are preferred as nucleic acid capturing agent according to the present invention. Silica wool made of silica fiber is particularly preferred.
Said nucleic acid purification method is characterized in that a branch for the washing solution flow path is provided in the flow path leading to the nucleic acid capturing tip, and washing solution is poured therein. The flow path can be shortened by switching the washing agent flow path. This makes it possible to reduce the size of the nucleic acid purification apparatus.
In said nucleic acid purification method, it is preferred to provide a special-purpose flow path to feed washing solution to the nucleic acid capturing tip. System operation and maintenance are facilitated by providing a simple washing solution flow path to the tip, without providing a washing solution switching flow path.
In said nucleic acid purification method, it is preferred to feed air into the flow path. This makes it possible to quickly remove from the tip the washing solution containing impurities included in nucleic acid sucked by the capturing agent.
In said nucleic acid purification method, it is preferred that discharge of washing solution and feed of air be repeated alternately. This ensures effective removal of washing solution remaining in the tip.
The present invention is characterized by a nucleic acid purification apparatus wherein a nucleic acid capturing tip incorporating the solid phase containing a nucleic acid capturing agent is used to allow said solid phase to capture a nucleic acid and to extract the nucleic acid;
said nucleic acid purification apparatus characterized in that washing solution is fed in the tip containing the solid phase capturing said nucleic acid unidirectionally from the head to the end.
In the above nucleic acid purification apparatus, it is preferred that the flow path leading to said nucleic acid capturing tip be provided with a branch for a washing solution flow path, and a means to supply washing solution be provided. The flow path can be shortened by switching the washing agent flow path. This makes it possible to reduce the size of the nucleic acid purification apparatus. In above nucleic acid purification method, it is preferred to provide a special-purpose flow path to feed washing solution to the nucleic acid capturing tip.
System operation and maintenance are facilitated by providing a simple washing solution flow path to the tip, without providing a washing solution switching flow path.
In above nucleic acid purification method, it is preferred to provide a means of feeding air into the flow path.
This makes it possible to quickly remove from the tip the washing solution containing impurities included in nucleic acid sucked by the capturing agent.
In said nucleic acid purification method, it is preferred that discharge of washing solution and feed of air be repeated alternately. This ensures effective removal of washing solution remaining in the tip.
The present invention is characterized by a nucleic acid purification apparatus wherein a nucleic acid capturing tip incorporating the solid phase containing a nucleic acid capturing agent is used to allow said solid phase to capture a nucleic acid and to extract the nucleic acid;
said nucleic acid purification apparatus comprising;
a nucleic acid capturing tip incorporating the solid phase containing a nucleic acid capturing agent,
a liquid suction/discharge movable nozzle in contact with said nucleic acid capturing tip in a removable manner,
a treatment solution capable of storing a mixture between the substance to promote capturing of nucleic acid by said solid phase and sample containing nucleic acid,
a means for feeding washing solution into the tip containing the solid phase capturing said nucleic acid unidirectionally from the head to the end,
a means for supplying eluent to said nucleic acid capturing tip,
a top removing means for removing said nucleic acid capturing tip from said liquid suction/discharge movable nozzle subsequent to discharge of eluent from said nucleic acid capturing tip to a purified product vessel, and
a purified product vessel to receive purified nucleic acid products.
The present invention provides a nucleic acid purification method and purification apparatus characterized by high washing efficiency where contamination does not occur and liquid does not remain in the nozzle tip.